Extended Data Fig. 3: Optimization and validation of ECM digestion and bcan deletion.
From: Extracellular matrix proteolysis maintains synapse plasticity during brain development
a) Representative images and quantification of hyaluronan depletion after hyaluronidase injection at 14 dpf, measured as mean fluorescence intensity of GFP (ubi:ssncan-GFP) at 6 h post injection (hpi) (vehicle, n = 7 fish; Hyal, n = 8 fish; p = 0.0023**, Welch’s t-test) and 24 hpi (vehicle, n = 5 fish; Hyal, n = 4 fish; p = 0.392, Welch’s t-test). GFP intensity normalized to mean of vehicle control. Dashed lines indicate hindbrain regions. Scale: 100 µm. b) Representative images and quantification of hyaluronan depletion at 6 hpi at 60 dpf, measured as mean fluorescence intensity of GFP (ubi:ssncan-GFP) nomalized to vehicle control (vehicle, n = 3 fish; Hyal, n = 3 fish; p = 0.049*, Welch’s t-test). Scale: 100 µm. c) Representative images and quantification of brevican depletion at 6 h after hyaluronidase injection at 14 dpf (vehicle, n = 16 fish; Hyal, n = 15 fish; p = 0.0018**, Welch’s t-test). Brevican intensity normalized to mean of vehicle control. Dashed lines indicate hindbrain regions. Scale: 20 µm. d) Representative images and quantification of SV2 presynapse marker 6 h after hyaluronidase injection at 14 dpf (vehicle, n = 7 fish; Hyal, n = 8 fish; p = 0.003**, Welch’s t-test). SV2 intensity normalized to mean of vehicle control. Scale: 100 µm. e) Non-merged images of time lapse imaging from Fig. 2d. Circles indicate stable synapses. Boxes indicate the region shown in Fig. 2d. Scale: 5 µm. f) Expression of bcan gene in bcan−/− fish assessed by RT-qPCR from whole larvae. Expression normalized to ef1a housekeeper gene (n = 3/group; p = 0.0064**, Welch’s t-test). g) Representative images and quantification of brevican protein in bcan−/− fish (bcan+/+, n = 10 fish; bcan−/−, n = 12 fish; p < 0.0001****, Welch’s t-test). Brevican intensity normalized to mean of bcan+/+ control. Scale: 100 µm. h) Representative images and quantification of SV2 presynapse marker in bcan−/− fish at 14 dpf (bcan+/+, n = 15 fish; bcan−/−, n = 15 fish, p = 0.049*, Welch’s t-test). SV2 intensity normalized to mean of bcan+/+ control. Scale: 100 µm. i) Non-merged images of time lapse imaging experiment from Fig. 2i. Circles indicate stable synapses. Boxes indicate the region shown in Fig. 2i. Scale: 5 µm. Values were plotted as mean ± SEM. ****: p < 0.0001; **: p < 0.01; *: p < 0.05; ns: not significant.
